Solid support with a grafted chain

ABSTRACT

Articles that contain a solid support with a grafted chain extending from the solid support, methods of making these articles, and various uses of the articles are described. More specifically, the grafted chain has a functional group that can react with or interact with target compound. Alternatively, the functional group on the grafted chain can react with a modifying agent to provide another group that can react with or interact with the target compound. The grafted chains are attached to the solid support through a ring-opened azlactone group. The articles can be used to purify the target compound or to separate the target compound from other molecules in a sample.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of pending prior U.S.application Ser. No. 14/254,247, filed Apr. 16, 2014, now allowed, whichis a divisional application of U.S. application Ser. No. 14/058,561,filed Oct. 21, 2013, now granted U.S. Pat. No. 8,735,455, which is adivisional application of U.S. application Ser. No. 13/000,435, filedDec. 21, 2010, now granted U.S. Pat. No. 8,592,493, which is a nationalstage filing under 35 U.S.C. §371 of International Application No.PCT/US2009/043455, filed May 11, 2009, which claims the benefit of U.S.Provisional Patent Application No. 61/075,934, filed Jun. 26, 2008, thedisclosures of which are incorporated by reference herein in theirentireties.

TECHNICAL FIELD

Articles that include a solid support and a grafted chain with afunctional group extending from the solid support, methods of makingthese articles, and various uses of these articles are described.

BACKGROUND

Various solid supports have been used for the separation and/orpurification of target compounds. For example, various polymeric solidsupports have been used to purify or separate target compounds based onthe presence of an ionic group, based on the size of the targetcompound, based on a hydrophobic interaction, based on an affinityinteraction, or based on the formation of a covalent bond.

In the biotechnology industry, large-scale separation and/orpurification of various biomolecules such as proteins, enzymes,vaccines, DNA, RNA, and the like are of great interest. Improvedmaterials and methods for separating and purifying biomolecules aredesired.

SUMMARY

Articles that contain a solid support with a grafted chain extendingfrom the solid support, methods of making these articles, and varioususes of the articles are described. More specifically, the grafted chainhas a functional group that can react with or interact with a targetcompound. Alternatively, the functional group on the grafted chain canreact with a modifying agent to provide another group that can reactwith or interact with the target compound. The grafted chains areattached to the solid support through a ring-opened azlactone group.

In a first aspect, a method of preparing an article is provided. Thearticle includes a solid support and a grafted chain extending from thesolid support. The method of preparing the article includes providing anazlactone-functionalized support of Formula (I).

In Formula (I), SS refers to a solid support, the variable p is aninteger equal to 0 or 1, and each R¹ is independently selected fromalkyl, heteroalkyl, aryl, or aralkyl. The method further includesforming a (meth)acryloyl-functionalized support of Formula (II)SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²═CH₂  (II)from the azlactone-functionalized support of Formula (I). In Formula(II), each Q is independently a divalent group selected from oxy, thio,or —NR³— where R³ is a hydrogen, alkyl, heteroalkyl, aryl, or aralkyl.Group Y¹ is a first linking group that contains an alkylene,heteroalkylene, arylene, or combination thereof. The group R² ishydrogen or an alkyl. The method still further includes reacting the(meth)acryloyl-functional support of Formula (II) with a monomercomposition that contains a monomer of Formula (III)Z¹—Y²—CR²═CH₂  (III)to form a grafted support of Formula (IV).SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²U²—CH₂—U¹  (IV)The group U¹ includes at least one divalent monomeric unit of formula—CR²(Y²Z¹)—CH₂—. Group U² is selected from hydrogen or a group thatincludes at least one divalent monomeric unit of formula—CR²(Y²Z¹)—CH₂—. The group Y² is a second linking group selected from asingle bond or a divalent group that contains an alkylene,heteroalkylene, arylene, or combination thereof. Group Z¹ is afunctional group selected from (1) an acidic group or a salt thereof,(2) an amino group or a salt thereof, (3) a hydroxyl group, (4) anazlactone group or a precursor of the azlactone group, (5) a glycidylgroup, or (6) a combination thereof.

In a second aspect, an article is provided that includes a graftedsupport of Formula (IV).SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²U²—CH₂—U¹  (IV)In Formula (IV), SS is a solid support and R¹ is each independentlyselected from alkyl, heteroalkyl, aryl, or aralkyl. The variable p is aninteger equal to 0 or 1. Each group Q is independently a divalent groupselected from oxy, thio, or —NR³— where R³ is hydrogen, alkyl,heteroalkyl, aryl, or aralkyl. Group Y¹ is a first linking group thatcontains an alkylene, heteroalkylene, arylene, or combination thereof.The group R² is hydrogen or an alkyl. The group U¹ includes at least onedivalent monomeric unit of formula —CR²(Y²Z¹)—CH₂—. Group U² is selectedfrom hydrogen or a group that includes at least one divalent monomericunit of formula —CR²(Y²Z¹)—CH₂—. The group Y² is a second linking groupselected from a single bond or a divalent group that contains analkylene, heteroalkylene, arylene, or combination thereof. Group Z¹ is afunctional group selected from (1) an acidic group or a salt thereof,(2) an amino group or a salt thereof, (3) a hydroxyl group, (4) anazlactone group or a precursor of the azlactone group, (5) a glycidylgroup, or (6) a combination thereof.

In a third aspect, a (meth)acryloyl-functionalized support of Formula(II) is provided.SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²═CH₂  (II)In Formula (II), SS is a solid support and R¹ is each independentlyselected from alkyl, heteroalkyl, aryl, or aralkyl. The variable p is aninteger equal to 0 or 1. Each Q is independently a divalent groupselected from oxy, thio, or —NR³— where R³ is hydrogen, alkyl,heteroalkyl, aryl, or aralkyl. Group Y¹ is a first linking group thatcontains an alkylene, heteroalkylene, arylene, or combination thereof.The group R² is hydrogen or an alkyl.

In a fourth aspect, a method of preparing an article is provided. Thearticle includes a solid support and a grafted chain extending form thesolid support. The method includes providing an azlactone-functionalizedsupport of Formula (I).

In Formula (I), SS refers to a solid support, the variable p is aninteger equal to 0 or 1, and each R¹ is independently selected fromalkyl, heteroalkyl, aryl, or aralkyl. The method further includesforming a (meth)acryloyl-functionalized support of Formula (II)SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²═CH₂  (II)from the azlactone-functionalized support of Formula (I). In Formula(II), each Q is independently a divalent group selected from oxy, thio,or —NR³— where R³ is a hydrogen, alkyl, heteroalkyl, aryl, or aralkyl.Group Y¹ is a first linking group that contains an alkylene,heteroalkylene, arylene, or combination thereof. The group R² ishydrogen or an alkyl. The method still further includes reacting the(meth)acryloyl-functional support of Formula (II) with a monomercomposition that contains a monomer of Formula (III)Z¹—Y²—CR²═CH₂  (III)to form a grafted support of Formula (IV).SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²U²—CH₂—U¹  (IV)The group U¹ includes at least one divalent monomeric unit of formula—CR²(Y²Z¹)—CH₂—. Group U² is hydrogen or is a group that includes atleast one divalent monomeric unit of formula —CR²(Y²Z¹)—CH₂—. The groupY² is a second linking group selected from a single bond or a divalentgroup that contains an alkylene, heteroalkylene, arylene, or combinationthereof. Group Z¹ is a functional group selected from (1) an acidicgroup or a salt thereof, (2) an amino group or a salt thereof, (3) ahydroxyl group, (4) an azlactone group or a precursor of the azlactonegroup, (5) a glycidyl group, or (6) a combination thereof. The methodyet further includes reacting the functional group Z¹ of the graftedsupport with a modifying agent of formula A-T to form a modified graftedsupport of Formula (V).SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²U⁴—CH2-U³  (V)In Formula (V), the group U³ includes at least one divalent monomericunit of formula —CR²(Y²-L-T)-CH₂—. Group U⁴ is selected from hydrogen ora group that includes at least one divalent monomeric unit of formula—CR²(Y²-L-T)-CH₂—. Group L is an attachment group formed by reactinggroup Z¹ on the grafted support with a modifying group A of themodifying agent. Group T is a remainder of the modifying agent A-T andis equal to the modifying agent A-T minus the modifying group A.

In a fifth aspect, an article is provided that includes a modifiedgrafted support of Formula (V).SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²U⁴—CH₂—U³  (V)In Formula (V), SS is a solid support and R¹ is each independentlyselected from alkyl, heteroalkyl, aryl, or aralkyl. The group R² ishydrogen or an alkyl. The variable p is an integer equal to 0 or 1. Eachgroup Q is independently a divalent group selected from oxy, thio, or—NR³— where R³ is hydrogen, alkyl, heteroalkyl, aryl, or aralkyl. Thegroup Y¹ is a first linking group containing an alkylene,heteroalkylene, arylene, or combination thereof. The group U³ includesat least one divalent monomeric unit of formula —CR²(Y²-L-T)-CH₂—. GroupU⁴ is selected from hydrogen or a group that includes at least onedivalent monomeric unit of formula —CR²(Y²-L-T)-CH₂—. Group L is anattachment group formed by reacting a group Z¹ with a modifying group Aof a modifying agent of formula A-T. Group T is a remainder of themodifying agent and is equal to the modifying agent A-T minus themodifying group A. Group Z¹ is a functional group selected from (1) anacidic group or a salt thereof, (2) an amino group or a salt thereof,(3) a hydroxyl group, (4) an azlactone group or a precursor of theazlactone group, (5) a glycidyl group, or (6) a combination thereof.

In a sixth aspect, a method of purifying or separating a target compoundis provided. The method includes providing an article that includes agrafted support of Formula (IV) as described above. The method furtherincludes contacting the article with a sample containing the targetcompound such that the target compound interacts with or reacts with atleast one functional group Z¹ on the grafted support.

In a seventh aspect, another method of purifying or separating a targetcompound is provided. The method includes providing an article thatincludes a modified grafted support of Formula (V) as described above.The method further includes contacting the article with a samplecontaining a target compound such that the target compound interactswith or reacts with at least one group T of the modified graftedsupport.

The above summary of the present invention is not intended to describeeach embodiment or every implementation of the present invention. TheDetailed Description and Examples that follow more particularlyexemplify these embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Articles that contain a solid support and a grafted chain extending fromthe solid support, methods of making these articles, and various uses ofthese articles are described. More specifically, the grafted chain has afunctional group that can react with or interact with a target compound.Alternatively, the functional group on the grafted chain can react witha modifying agent to provide another group that can react with orinteract with the target compound. The reaction or interaction with thetarget compound can be used, for example, to purify the target compoundor to separate the target compound from other molecules in a sample. Inat least some embodiments, the binding capacity for the target compoundcan be improved through the positioning of the functional group on thegrafted chain rather than on a surface of the solid support.

As used herein, the terms “a”, “an”, and “the” are used interchangeablywith “at least one” to mean one or more of the elements being described.

The term “azlactone group” refers to a monovalent group of formula

where each R¹ is independently selected from alkyl, heteroalkyl, aryl,or aralkyl and the variable p is selected from zero or one. In manyembodiments, R¹ is methyl. The azlactone group may be referred to as“Az” herein.

The term “precursor of the azlactone group” refers to a group that canundergo a ring closure reaction to form the azlactone group and is offormula —(CO)—NH—C(R¹)₂—(CH₂)_(p)—COOH or a salt thereof where R¹ and pare defined above for the azlactone group. For example,N-acryloylmethylalanine can be polymerized and then subjected to a ringclosure reaction to provide a polymeric material with azlactone groups.In N-acryloylmethylalanine, the precursor of the azlactone group is—(CO)—NH—C(CH₃)₂—COOH or a salt thereof.

The term “alkyl” refers to a monovalent hydrocarbon group that issaturated and has up to 18 carbon atoms. The alkyl group can be linear,branched, cyclic, or a combination thereof. In some examples, the alkylgroup is linear or branched and has 1 to 12 carbon atoms, 3 to 12 carbonatoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 3to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In otherexamples, the alkyl is cyclic (i.e., the alkyl is a cycloalkyl) and has3 to 12 carbon atoms, 3 to 6 carbon atoms, or 4 to 6 carbon atoms.

The term “alkylene” refers to a divalent hydrocarbon group that issaturated and that has up to 18 carbon atoms. The alkylene group can belinear, branched, cyclic, or a combination thereof. In some examples,the alkylene group is linear or branched and has 1 to 12 carbon atoms, 1to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4carbon atoms, or 1 to 3 carbon atoms. In other examples, the alkylene iscyclic (i.e., the alkyl is a cycloalkyl) and has 3 to 12 carbon atoms, 3to 6 carbon atoms, or 4 to 6 carbon atoms.

The term “heteroalkyl” refers to a monovalent group that is saturatedand that has at least two carbon atoms separated by at least onecatenary heteroatom selected from oxygen (i.e., oxy), sulfur (i.e.,thio), or —NR³— where R³ is hydrogen, alkyl, heteroalkyl, aryl, oraralkyl. The heteroalkyl group can be linear, branched, cyclic, or acombination thereof. In some examples, the heteroalkyl group has 2 to 18carbon atoms and 1 to 6 heteroatoms, 2 to 12 carbon atoms and 1 to 6heteroatoms, 2 to 10 carbon atoms and 1 to 5 heteroatoms, 2 to 8 carbonatoms and 1 to 4 heteroatoms, or 2 to 6 carbon atoms and 1 to 3heteroatoms.

The term “heteroalkylene” refers to a divalent group that is saturatedand that has at least two carbon atoms separated by at least onecatenary heteroatom selected from oxygen (i.e., oxy), sulfur (i.e.,thio), or —NR³— where R³ is hydrogen, alkyl, heteroalkyl, aryl, oraralkyl. The heteroalkylene group can be linear, branched, cyclic, or acombination thereof. In some examples, the heteroalkylene group has 2 to18 carbon atoms and 1 to 6 heteroatoms, 2 to 12 carbon atoms and 1 to 6heteroatoms, 2 to 10 carbon atoms and 1 to 5 heteroatoms, 2 to 8 carbonatoms and 1 to 4 heteroatoms, or 2 to 6 carbon atoms and 1 to 3heteroatoms.

The term “aryl” refers to a heterocyclic or carbocyclic monovalentaromatic group. An aryl can have one or more connected or fused rings.Some exemplary aryl groups have a 5 to 12 membered ring structure with 0to 3 heteroatoms selected from oxygen (i.e., oxy), sulfur (i.e., thio),or —NR³— where R³ is hydrogen, alkyl, heteroalkyl, aryl, or aralkyl. Forexample, the aryl group can have 2 to 12 carbon atoms and 0 to 3heteroatoms, 3 to 12 carbon atoms and 0 to 2 heteroatoms, or 4 to 12carbon atoms and 0 to 1 heteroatoms.

The term “arylene” refers to a heterocyclic or carbocyclic divalentaromatic group. An arylene can have one or more connected or fusedrings. Some exemplary arylene groups have a 5 to 12 membered ringstructure with 0 to 3 heteroatoms selected from oxygen (i.e., oxy),sulfur (i.e., thio), or —NR³— where R³ is hydrogen, alkyl, heteroalkyl,aryl, or aralkyl.

The term “aralkyl” refers to an alkyl group that is substituted with anaryl group. An aralkyl group can have, for example, 3 to 15 carbon atomsand 0 to 3 heteroatoms, 4 to 15 carbon atoms and 0 to 2 heteroatoms, or5 to 15 carbon atoms and 0 to 1 heteroatoms.

The term “carbonyl” refers to a divalent group of formula —(CO)— with adouble bond between the carbon and oxygen.

The term “carbonyloxy” refers to a divalent group of formula —(CO)—O—where (CO) refers to a carbonyl group.

The term “carbonylthio” refers to a divalent group of formula —(CO)—S—where (CO) refers to a carbonyl group.

The term “carbonylimino” refers to a divalent group of formula—(CO)—NR³— where R³ is hydrogen, alkyl, heteroalkyl, aryl, or aralkyl.

The term “glycidyl” refers to a monovalent group of formula

where each R⁴ is independently a hydrogen, alkyl, heteroalkyl, aryl, oraralkyl. In many glycidyl groups, each R⁴ is hydrogen.

The term “(meth)acryloyl” refers to a group of formula H₂C═CR^(b)—(CO)—where R^(b) is hydrogen or alkyl (e.g., methyl).

The term “(meth)acrylamido” refers to a group of formulaH₂C═CR^(b)—(CO)—NR^(a)— where R^(b) is hydrogen or alkyl (e.g., methyl)and R^(a) is hydrogen, alkyl, heteroalkyl, aryl, or aralkyl.

The term “(meth)acryloxy” refers to a group of formulaH₂C═CR^(b)—(CO)—O— where R^(b) is hydrogen or alkyl (e.g., methyl).

The term “nucleophilic group” refers to a hydroxyl group (i.e., —OH),thiol group (i.e., —SH), primary amino group (i.e., —NH₂), or secondaryamino group of formula —NHR^(a) wherein R^(a) is an alkyl, heteroalkyl,aryl, or aralkyl.

The terms “polymer” or “polymeric” refer to a material that is ahomopolymer, copolymer, terpolymer, or the like. Likewise, the terms“polymerize” or “polymerization” refer to the process of making ahomopolymer, copolymer, terpolymer, or the like.

The phrase “in the range of” includes the endpoints of the range and allthe numbers between the endpoints. For example, the phrase in the rangeof 1 to 10 includes 1, 10, and all numbers between 1 and 10. Further,any recitation of a range that is not specifically called a rangeincludes the endpoint and all number between the endpoints unlessspecifically stated otherwise.

The phrase “and/or” means either of the options listed or both of theoptions listed. For example, the expression A and/or B means A alone, Balone, or both A and B.

The articles are prepared from an azlactone-functionalized support,which is a solid support having at least one azlactone group on asurface of the solid support. Any known azlactone-functionalized supportcan be used. At least one azlactone group of theazlactone-functionalized support is reacted to form a(meth)acryloyl-functionalized support. The (meth)acryloyl group of the(meth)acryloyl-functional functionalized support can be polymerized withother ethylenically unsaturated monomers to form the grafted chains. Thegrafted chains are covalently attached to the solid support through aring opened azlactone group. The grafted chains include a functionalgroup selected from (1) an acidic group or a salt thereof, (2) an aminogroup or a salt thereof, (3) a hydroxyl group, (4) an azlactone group ora precursor of the azlactone group, (5) a glycidyl group, or (6) acombination thereof. In some embodiments, the functional group iscapable of reacting with or interacting with a target compound. In otherembodiments, the functional group is modified to form another group thatis capable of reacting with or interacting with the target compound. Thefunctional group is modified by reacting with a modifying agent.

The azlactone-functionalized support is of Formula (I).

In Formula (I), SS refers to a solid support, the variable p is aninteger equal to zero or one, and each R¹ is independently selected fromalkyl, heteroalkyl, aryl, or aralkyl. Although Formula (I) shows onlyone azlactone group attached to the solid support for ease ofdescription, multiple azlactone groups are typically attached to thesolid support. The azlactone-functionalized support of Formula (I) canbe referred to herein with the formula SS-Az.

The solid support with at least one azlactone group on its surface(i.e., an azlactone-functionalized support) can be in the form of abead, membrane, foam, film, sheet, coating on a substrate, or the like.Azlactone-functionalized supports and methods of making such supportsare described, for example, in U.S. Pat. No. 5,336,742 (Heilmann etal.), U.S. Pat. No. 5,403,902 (Heilmann et al.), U.S. Pat. No. 5,344,701(Gagnon et al.), U.S. Pat. No. 5,993,935 (Rasmussen et al.), U.S. Pat.No. 6,063,484 (Exsted et al.), U.S. Pat. No. 5,292,514 (Capecchi etal.), U.S. Pat. No. 6,548,607 (Halverson et al.), U.S. Pat. No.5,408,002 (Coleman et al.), U.S. Pat. No. 5,476,665 (Dennison), U.S.Pat. No. 5,510,421 (Dennison et al.), and U.S. Pat. No. 6,794,458(Haddad et al.).

Suitable azlactone-functionalized supports can be prepared using avariety of methods. In some methods, the azlactone-functionalizedsupport can be prepared using reverse phase suspension polymerization, atechnique in which the polymerization reaction occurs within waterdroplets suspended in the suspending medium. The suspending medium iswater immiscible and the monomers are water-soluble.

In one reverse phase polymerization process, the polymerization mediumincludes at least one alkenyl azlactone and at least one crosslinkingmonomer in a water miscible cosolvent. The amount of crosslinkingaffects polymeric properties such as the porosity and the degree ofswelling in a solvent. Suitable alkenyl azlactone monomers include, butare not limited to, 2-vinyl-4,4-dimethyl-2-oxazolin-5-one which iscommercially available from SNPE, Inc., Princeton, N.J.;2-isopropenyl-4,4-dimethyl-2-oxazolin-5-one; and2-vinyl-4,4-dimethyl-1,3-oxazin-6-one. Suitable crosslinking agentsinclude, but are not limited to, ethylenically unsaturated(α,β-unsaturated) esters such as ethylene (meth)diacrylate andtrimethylolpropane tri(meth)acrylate; and ethylenically unsaturatedamides such as methylenebis(meth)acrylamide andN,N′-di(meth)acryloyl-1,2-diaminoethane. Additionally, thepolymerization medium can include other monomers that are water solubleand that can be polymerized using a free radical addition polymerizationreaction. Suitable optional monomers include, for example,N,N-dimethylacrylamide and N-vinylpyrrolidone. This polymerizationprocess is further described in U.S. Pat. No. 5,403,902 (Heilmann etal.) and U.S. Pat. No. 5,336,742 (Heilmann et al.)

In another reverse phase polymerization process, a two-steppolymerization process is used to prepare azlactone-functionalizedsupports. In a first step, polymeric material is prepared that hascarboxylic functional groups. The carboxylic functional groups aresubsequently reacted with a cyclization agent to form azlactone groups.The polymerization medium includes a water-soluble salt of aN-(meth)acryloylamino acid, a crosslinking monomer, and a waterimmiscible suspending medium. Additionally, the polymerization mediumcan include other monomers that are water soluble and that can bepolymerized using a free radical addition polymerization reaction.Suitable optional monomers include, for example, N,N-dimethylacrylamideand N-vinylpyrrolidone. Suitable cyclization agents include, forexample, acetic anhydride, trifluoroacetic anhydride, and alkylchloroformates. This process is further described in U.S. Pat. No.5,403,902 (Heilmann et al.) and U.S. Pat. No. 5,336,742 (Heilmann etal.).

In other methods, the azlactone-functionalized support can be preparedusing dispersion polymerization, a technique in which a dispersingmedium is chosen that will dissolve the monomers but that willprecipitate the polymer as it forms. Various surfactants can be added toprevent aggregation of the polymer particles. For example, theazlactone-functionalized support can be prepared using a dispersionpolymerization process in which the polymerization medium includes a2-alkenyl azlactone monomer, a crosslinking monomer, and at least onesurfactant in an organic solvent such as an alcohol. This process isfurther described in U.S. Pat. No. 5,403,902 (Heilmann et al.) and U.S.Pat. No. 5,336,742 (Heilmann et al.).

The polymeric azlactone-functionalized support can be a gel-type ormacroporous polymeric material. As used herein, the term “gel-type”refers to a polymeric material that is prepared with less than 20 weightpercent crosslinker based on the weight of monomers in thepolymerization medium. As used herein, the term “macroporous” refers toa polymeric material that is prepared with at least 20 weight percentcrosslinker based on the weight of monomers in the polymerizationmedium. A gel-type material tends to swell more and tends to be lessrigid than macroporous materials.

In some embodiments, the substrate is the form of a bead. The beads canhave a spherical shape, regular shape, or irregular shape. Beads can beprepared using either reverse phase suspension polymerization techniquesor dispersion polymerization techniques. Beads that are prepared usingreverse phase suspension polymerization techniques tend to be moreporous and to have larger surface areas compared to beads that areprepared using dispersion polymerization techniques. Beads preparedusing dispersion polymerization techniques are generally smaller in sizeand are less porous (e.g., in some cases the beads can be substantiallynonporous) than beads that are prepared using reverse phase suspensionpolymerization techniques.

The size of the beads can vary depending on the particular application.Generally, the average diameter of the beads is in the range of 0.1micrometers to 5 millimeters. Some exemplary beads have an averagediameter of 0.1 to 1,000 micrometers, 0.1 to 500 micrometers, 0.1 to 100micrometers, 0.5 to 100 micrometers, 0.1 to 50 micrometers, 0.1 to 20micrometers, 0.1 to 3 micrometers, or 0.5 to 3 micrometers.

In some methods of making an azlactone-functionalized support, thesubstrate is in the form of a composite membrane that includesazlactone-containing particles (e.g., beads) dispersed in a continuous,porous matrix. Such composite membranes are further described in U.S.Pat. No. 5,993,935 (Rasmussen et al.). The azlactone-containingparticles included in the composite membrane can be the beads describedabove. Alternatively, the azlactone-containing particles included in themembranes can be inorganic particles modified with a coating compositionto provide a surface with azlactone groups. The inorganic particles cancontain, for example, metals or metal oxides, ceramic materials such asalumina, silica, zirconia, or mixtures thereof, glass (e.g., beads orbubbles), controlled pore glass, and the like. These particles can bemodified by coating the particles with a polymer that contains reactiveazlactone functional groups or by reacting groups on the surface of theparticles with a reagent that contains a reactive functional group(e.g., a coupling agent that has an alkoxy silane for reacting with thesurface of the inorganic particle and that also contains an azlactonegroup).

Useful continuous, porous matrices for the composite membrane include,but are not limited to, woven and nonwoven fibrous webs or porousfibers. Exemplary fibrous materials include those fabricated frompolyolefins (e.g., polyethylene and polypropylene), polyvinyl chloride,polyamides (e.g., nylons), polystyrenes, polysulfones, polyvinylalcohol, polybutylene, ethyl vinyl acetate, poly(meth)acrylates such aspolymethyl methacrylate, polycarbonate, cellulosics (e.g., celluloseacetate), polyesters (e.g., polyethylene terephthalate), polyimides,polyurethanes (e.g., polyether polyurethanes), and combinations thereof.

In another method of preparing a composite membrane,azlactone-containing particles are dispersed in a liquid to form acolloidal suspension. A thermoplastic polymer is melt blended with thecolloidal suspension at a temperature sufficient to form a homogenoussolution. The solution can be formed into a desired shape and thencooled to induce phase separation of the liquid from the polymericmaterial and solidify the polymeric material. After removal of theliquid, the azlactone-containing particles are dispersed in amicroporous polymer matrix. This method is described in detail in U.S.Pat. No. 4,957,943 (McAllister et al.).

The composite membranes can also be prepared from a porous fibrillatedpolymer matrix such as fibrillated polytetrafluoroethylene (PTFE). Theazlactone-containing particles can be blended with a PTFE dispersion toobtain a putty-like mass. The putty-like mass can then be mixed at atemperature between 5° C. and 100° C. to cause fibrillation of the PTFEand biaxially calendered to form a sheet. The sheet can be dried toremove any solvent. Such methods of making membranes are furtherdescribed in U.S. Pat. No. 4,153,661 (Ree et al.); U.S. Pat. No.4,565,663 (Errede et al.); U.S. Pat. No. 4,810,381 (Hagen et al.); andU.S. Pat. No. 4,971,736 (Hagen et al.).

Yet another method of making a composite membrane is described in U.S.Pat. No. 4,539,256 (Shipman). Azlactone-containing particles can bedispersed in a polyolefin by heating and stirring. The resulting moltenmixture is cast onto a heated plate, subjected to pressure, and thencooled in ice water.

Additionally, composite membranes can also be formed using solvent phaseinversion techniques as described in U.S. Pat. No. 5,476,665 (Dennison).An azlactone-containing particle and blending polymers are introducedinto a vessel containing a solvent capable of dissolving the polymers,casting the solution into a desired shape, and introducing the castshape to a coagulation bath of a liquid that is miscible with thesolvent but in which the polymers precipitate to form anazlactone-functionalized membrane.

Azlactone-functionalized supports can also be formed from polymer blendsas described in U.S. Pat. No. 5,408,002 (Coleman et al.) and U.S. Pat.No. 6,063,484 (Exsted et al.). Azlactone-containing homopolymersprepared from 2-alkenyl azlactone can be melt blended with thermoplasticpolymers. Suitable thermoplastics include polyamides (e.g., nylon 6),polyurethanes, poly(meth)acrylates, polystyrene, polyolefins, ethylenevinyl acetate copolymers, poly(N-vinyl lactams) (e.g., polyvinylpyrrolidone), polyvinyl acetate, polyoxyalkylene oxides,fluoroelastomers, polycarbonates, polyesters, and the like.

Another method of preparing azlactone-functionalized substrates isdescribed in U.S. Pat. No. 6,063,484 (Exsted et al.). A polyolefin resinis mixed with a free radical initiator (e.g., a peroxide or azocompound) and then heated in an extruder at a temperature sufficient togenerate free radicals. A 2-alkenyl azlactone is injected into theextruder to form a grafted azlactone thermoplastic composition. Thiscomposition is then formed into a membrane.

Alternatively, azlactone-functionalized supports can be formed usingsolvent phase inversion of an azlactone-containing polymer as describedin U.S. Pat. No. 5,510,421 (Dennison et al.). Azlactone-containingcompositions and blending polymers are placed in a vessel containing asolvent capable of dissolving them. The solution is then cast into asuitable shape, which is then introduced into a coagulation bath of aliquid miscible with the solvent but that causes the precipitation of anazlactone-functionalized membrane.

An azlactone-functionalized support can also be prepared by applying acoating composition to a solid support. Exemplary solid supports can beprepared from metal, metal oxide or hydroxide, polymeric material,glass, ceramic material, or a combination thereof. The solid support canhave any desired shape or size. For example, the supports can be films,particles, fibers, woven or nonwoven webs, membranes, molded plasticarticles, and the like. In some embodiments, the coating composition caninclude a soluble polymer having azlactone groups (e.g., a polymerformed by free radical polymerization of an alkenyl azlactone monomer)and a crosslinking agent. The coating composition can be applied to thesolid support using techniques such as extrusion coating, die coating,dip coating, air-knife coating, gravure coating, curtain coating, spraycoating, and the like. This process is further described in U.S. Pat.No. 6,794,458 (Haddad et al.). In other embodiments, a surface of asolid support is coated with a coating composition that includesazlactone-functional monomers and crosslinking monomers. The coatingcomposition is polymerized to form an azlactone-functional surface layeron the solid support. This embodiment is further described in U.S. Pat.No. 5,344,701 (Gagnon et al.).

In some examples, there is sufficient adhesion of the coatingcomposition containing the soluble polymer having azlactone groups tothe surface of the solid support. With other solid supports, theadhesion can be enhanced by various pretreatments such as plasma orcorona treatment of the solid support or by using a primer layer betweenthe solid support and the coating composition.

Polymeric beads with azlactone groups are commercially available underthe trade designation “EMPHAZE” from 3M Company, St. Paul, Minn.

The azlactone-functionalized support of Formula (I) is reacted to form aa (meth)acryloyl-functionalized support of Formula (II).SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²═CH₂  (II)In Formula (II), SS refers to the solid support. Group R¹ and variable pare the same as described above for Formula (I). Each group Q isindependently a divalent group selected from oxy, thio, or —NR³— whereR³ is a hydrogen, alkyl, heteroalkyl, aryl, or aralkyl. Group R² istypically hydrogen or an alkyl. Group Y¹ in Formula (II) is a firstlinking group that contains an alkylene, heteroalkylene, arylene, orcombination thereof. The group Y¹ can further contain other optionalgroups that link together two or more alkylene groups, heteroalkylenegroups, arylene groups, or combinations thereof. The optional groups caninclude, for example, a carbonyl, carbonyloxy, carbonylthio,carbonylimino, oxy, thio, —NR³—, or combination thereof.

The (meth)acryloyl-functionalized support of Formula (II) can beprepared from the azlactone-functionalized support of Formula (I) usingany known synthesis method. In one exemplary method, theazlactone-functionalized support of Formula (I) is reacted with acompound having both (a) a nucleophilic group and (b) a (meth)acryloylgroup. For example, the compound can be of Formula (VI).HQ-Y¹-Q-(CO)—CR²═CH₂  (VI)In Formula (VI), Y¹ and Q are the same as described above for Formula(II). The group -QH, which corresponds to —OH, —SH, or —NR³H, is thenucleophilic group that can react with an azlactone group of theazlactone-functionalized support. This reaction results in the openingof the azlactone ring.

Exemplary compounds of Formula (VI) where the group -QH is a hydroxylgroup include, but are not limited to, hydroxyalkyl (meth)acrylates suchas 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl(meth)acrylate,3-hydroxypropyl(meth)acrylate, 4-hydroxybutyl(meth)acrylate,glycerol(meth)acrylate, and glycerol di(meth)acrylate; andhydroxyalkyl(meth)acrylamides such as hydroxypropyl(meth)acrylamide andmethylol(meth)acrylamide.

Exemplary compounds of Formula (VI) where the group -QH is a primary orsecondary amino group include, but are not limited to,aminoalkyl(meth)acrylamides such as aminoethyl(meth)acrylamide andaminopropyl(meth)acrylamide; N-alkylaminoalkyl(meth)acrylamides such asN-methylaminoethyl(meth)acrylamide andN-isopropylaminopropyl(meth)acrylamide; aminoalkyl(meth)acrylates suchas aminoethyl(meth)acrylate, aminopropyl(meth)acrylate; andN-alkylaminoalkyl(meth)acrylates such asN-methylaminoethyl(meth)acrylate and N-methylaminopropyl(meth)acrylate.

The reaction of the azlactone-functionalized support of Formula (I) withthe compound of Formula (VI) is shown in Reaction Scheme A where thegroup “Az” represents an azlactone group and the group “Az¹” representsa ring-opened azlactone group. As used herein, the ring-opened azlactonegroup Az¹ is a divalent group of formula—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)—. The (meth)acryloyl-functionalizedsupport of Formula (II) can be written using the formulaSS-Az¹-Q-Y¹-Q-(CO)—CR²═CH₂.

Reaction Scheme A

SS-Az+HQ-Y¹-Q-(CO)—CR²═CH₂→SS-Az¹-Q-Y¹-Q-(CO)—CR²═CH₂

In another exemplary method of preparing the(meth)acryloyl-functionalized support of Formula (II), theazlactone-functionalized support of Formula (I) is reacted with anucleophilic compound having at least two nucleophilic groups selectedfrom a hydroxyl group (—OH), thiol group (—SH), primary amino group(—NH₂), or secondary amino group (—NHR³). A first nucleophilic group canreact with an azlactone group of the azlactone-functionalized support ofFormula (I). This reaction opens the azlactone ring. The secondnucleophilic group can react with another compound such that the producthas a (meth)acryloyl group. The compound having two nucleophilic groupscan be represented by Formula (VII).HQ-Y^(1a)-QH  (VII)In Formula (VII), Q is the same as defined previously for Formula (II).Like group Y¹, the group Y^(1a) includes an alkylene, heteroalkylene,arylene, or combination thereof and can optionally further include anoxy, thio, amino (—NR³—), carbonyloxy, carbonylthio, carbonylimino, orcombination thereof.

Some exemplary nucleophilic compounds of Formula (VII) are alcoholamines (i.e., hydroxyamines) having both a hydroxyl group and a primaryor secondary amino group. Exemplary alcohol amines include, but are notlimited to, 2-hydroxyethylamine, 3-hydroxypropylamine,4-hydroxybutylamine, 1,2-dihydroxy-3-aminopropane,1-hydroxy-6-aminohexane, bis-(2-hydroxyethyl)amine, triethanolamine,1-amino-3,5-dihydroxycyclohexane, 1-amino-3,5-dihydroxybenzene,N,N′-bis(2-hydroxyethyl)piperazine, N-hydroxyethylpiperazine,2-amino-2-methyl-1,3-propanediol, and the like. Other exemplarynucleophilic compounds of Formula (VI) have at least two primary orsecondary amino groups such as hydrazine, adipic dihydrazide,ethylenediamine, N-methylethylenediamine, piperazine,N-(2-aminoethyl)-piperazine, 1,3-propanediamine, 1,4-butanediamine,benzenediamine, m-xylylenediamine, 1,3-cyclohexane-bis-methylamine,1,4-cyclohexanediamine, 1,8-diamino-3,6-dioxaoctane,1,3-diamino-2-hydroxypropane, tris-(2-aminoethyl)amine, and1,6-hexanediamine. Additional nucleophilic compounds with at least twoamino groups are polyether amines such as those commercially availableunder the trade designation JEFFAMINE from Huntsman Corporation (TheWoodlands, Tex.). Still other exemplary nucleophilic compounds ofFormula (VI) are compounds with two or more hydroxyl groups such asethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol,1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol,glycerol, trimethylolpropane, pentaerythritol, dihydroxybenzene,trihydroxybenzene, and bisphenol A. Additional nucleophilic compoundswith at least two hydroxyl groups are polyethylene oxides andpolypropylene oxides having hydroxyl end groups.

The reaction of the azlactone-functionalized support of Formula (I) witha nucleophilic compound of Formula (VII) results in the opening of theazlactone ring as shown in the intermediate of Formula (VIII)SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y^(1a)-QH  (VIII)that can also be represented by the formula SS-Az¹-Q-Y^(1a)-QH. Thisintermediate of Formula (VIII) can then be reacted with a secondcompound such that the product has a (meth)acryloyl group. The secondcompound can have a first group capable of reacting with thenucleophilic group -QH of the intermediate and a second group that isethylenically unsaturated. For example, the intermediate can react witha second compound that is a vinyl azlactone (e.g., vinyl dimethylazlactone), acyl halide having a (meth)acryloyl group, anhydride havinga (meth)acryloyl group, isocyanatoalkyl(meth)acrylate such asisocyanatoethyl(meth)acrylate, or glycidyl(meth)acrylate. This method isexemplified in Reaction Scheme B for a second compound that is a vinylazlactone of formula Az-CR²═CH₂.

Reaction Scheme B

SS-Az+HQ-Y^(1a)-QH→SS-Az¹-Q-Y^(1a)-QHSS-Az¹-Q-Y^(1a)-QH+Az-CR²═CH₂→SS-Az¹-Q-Y^(1a)-Q-Az¹-CR²═CH₂The formula SS-Az¹-Q-Y^(1a)-Q-Az¹-CR²═CH₂ corresponds toSS-Az¹-Q-Y^(1a)-Q-(CO)—(CH₂)_(p)—C(R¹)₂—NH—(CO)—CR²═CH₂, which in turncorresponds to Formula (II) where Y¹ is equal to—Y^(1a)-Q-(CO)—CH₂)_(p)—C(R¹)₂— and where one of the Q groups is anamino group.

The (meth)acryloyl-functionalized support of Formula (II) can then bereacted with a monomer composition that contains a functional monomer ofFormula (III).Z¹—Y²—CR²═CH₂  (III)In Formula (III), group Y² is a second linking group selected from asingle bond or a divalent group that contains an alkylene,heteroalkylene, arylene, or combination thereof. The group Y² canfurther contain other optional groups that function to connect two ormore alkylenes, heteroalkylenes, arylenes, or mixtures thereof. Theoptional groups can include, for example, a carbonyl, carbonyloxy,carbonylthio, carbonylimino, oxy, thio, —NR³—, or combination thereof.Group Z¹ is a first functional group selected from (1) an acidic groupor a salt thereof, (2) an amino group or a salt thereof, (3) a hydroxylgroup, (4) an azlactone group or a precursor to the azlactone group, (5)a glycidyl group, or (6) a combination thereof. Groups R² and R³ are thesame as described above for Formula (II).

The functional monomer of Formula (III) has both an ethylenicallyunsaturated group capable of undergoing a free radical polymerizationreaction plus a functional group Z¹. The functional monomer of Formula(III) undergoes a free radical polymerization reaction with the(meth)acryloyl-functionalized support of Formula (II) resulting in theformation of a grafted support that includes a grafted chain extendingfrom the solid support. In many embodiments, the solid support hasmultiple grafted chains. The grafted chain includes at least onedivalent monomeric unit of formula —CR²(Y²Z¹)—CH₂—. The grafted chain isoften polymeric and contains at least two or at least three monomericunits of formula —CR²(Y²Z¹)—CH₂—. The grafted chains have at least onependant group of formula —Y²—Z¹. If the grafted chain is polymeric, thegrafted chain contains at least two or at least three pendant groups offormula —Y²Z¹.

In some embodiments, the functional monomer of Formula (III) has a(meth)acrylolyl group as shown in Formula (IIIa).Z¹—Y^(2a)-Q-(CO)—CR²═CH₂  (IIIa)The group Y^(2a) is a divalent group that includes an alkylene,heteroalkylene, arylene, or combination thereof and optionally caninclude an oxy, thio, amino, carbonylimino, carbonyloxy, carbonylthio,or a combination thereof.

The group Z¹ of the functional monomers of Formula (III) or (IIIa) canbe an acidic group or a salt thereof. The functional monomer can be aweak acid, a salt of a weak acid, a strong acid, a salt of a strongacid, or a combination thereof. The functional monomer can be in aneutral state but capable of being negatively charged if the pH isadjusted. Some exemplary functional monomers of Formulas (III) or (IIIa)are sulfonic acids or salts thereof such as (meth)acrylamidosulfonicacids or salts thereof. More specific (meth)acrylamidosulfonic acidsinclude, but are not limited to, N-(meth)acrylamidomethanesulfonic acid,2-(meth)acrylamidoethanesulfonic acid, and2-(meth)acrylamido-2-methylpropanesulfonic acid. Salts of these acidicmonomer can also be used. Some other exemplary functional monomershaving an acid group or salt thereof include other sulfonic acids suchas vinylsulfonic acid, 3-sulfopropyl(meth)acrylate,sulfoethyl(meth)acrylate, and 4-styrenesulfonic acid.

Still other exemplary functional monomers having an acid group include,but are not limited to, phosphonic acids or salts thereof or carboxylicacids or salts thereof. For example, the function monomers can be(meth)acrylamidoalkylphosphonic acids such as2-(meth)acrylamidoethylphosphonic acid and3-(meth)acrylamidopropylphosphonic acid; acrylic acid and methacrylicacid; and carboxyalkyl (meth)acrylates such as 2-carboxyethyl(meth)acrylate and 3-carboxypropyl (meth)acrylate. Still other suitablemonomers include (meth)acryloylamino acids, such as those described inU.S. Pat. No. 4,157,418 (Heilmann). Exemplary (meth)acryloylamino acidsinclude, but are not limited to, N-(meth)acryloylglycine,N-(meth)acryloylaspartic acid, N-(meth)acryloyl-β-alanine, andN-(meth)acryloyl-2-methylalanine. Salts of any of these acidic monomerscan also be used. If the functional monomer is in the form of a salt ofa weak acid or a salt of a strong acid, the counter ion of these saltscan be, but are not limited to, alkali metal ions, alkaline earth metalions, ammonium ions, or tetraalkylammonium ions.

A second type of functional monomer of Formula (III) or (IIIa) has anamino group or a salt thereof for Z¹. The amino group or salt thereofcan be a primary amino group, secondary amino group, tertiary aminogroup, or quaternary ammonium group. This type of functional monomer canbe a weak base, a strong base, a salt of a weak base, a salt of a strongbase, or a mixture thereof. The functional monomer can be in a neutralstate but capable of being positively charged if the pH is adjusted. Ifthe functional monomer is in the form of a salt, the counter ion can be,but is not limited to, a halide (e.g., chloride), a carboxylate (e.g.,acetate or formate), nitrate, phosphate, sulfate, bisulfate, methylsulfate, or hydroxide.

Some exemplary functional monomers having an amino group or salt thereofinclude amino(meth)acrylates or amino(meth)acrylamides (as well asquaternary ammonium salts thereof) as shown in Formula (IIIb)N(R⁵)_(u)—Y^(2a)-Q-(CO)—CR²═CH₂  (IIIb)In Formula (IIIb), Y^(2a), Q, and R² are the same as described above forFormula (III) or (IIIa). Each R⁵ is independently hydrogen, alkyl,hydroxyalkyl (i.e., an alkyl substituted with a hydroxy), aminoalkyl(i.e., an alkyl substituted with an amino), aryl, or aralkyl. Thevariable u is equal to 2 for a primary, secondary, or tertiary aminogroup and equal to 3 for quaternary amino group. When u is equal to 3,the three R⁵ groups are independently selected from alkyl, hydroxyalkyl,aminoalkyl, aryl, or arylalkyl. That is, R⁵ typically is not equal tohydrogen when the variable u is equal to 3.

When the variable u is equal to 2, the R⁵ groups in Formula (IIIb) takentogether with the nitrogen atom to which they are attached can form aheterocyclic group that is aromatic, partially unsaturated (i.e.,unsaturated but not aromatic), or saturated. Such a heterocyclic groupcan optionally be fused to a second ring that is aromatic (e.g.,benzene), partially unsaturated (e.g., cyclohexene), or saturated (e.g.,cyclohexane). The counter ions of the quaternary ammonium salts areoften halides, sulfates, phosphates, nitrates, and the like.

In some embodiments of Formula (IIIb) where the variable u is equal to2, both R⁵ groups are hydrogen. In other embodiments where the variableu is equal to 2, one R⁵ group is hydrogen and the other is an alkylhaving 1 to 10, 1 to 6, or 1 to 4 carbon atoms. In still otherembodiments where the variable u is equal to 2, at least one of R⁵groups is a hydroxyl alkyl or an amino alkyl that has 2 to 10, 2 to 6,or 2 to 4 carbon atoms with the hydroxyl or amino group positioned onany of the carbon atoms of the alkyl group except the first. In stillother embodiments where the variable u is equal to 2, at least one ofthe R⁵ groups is an aryl having 5 or 6 carbon atoms; or an aralkyl withthe alkyl group having 1 to 10 carbon atoms and the aryl group having 5or 6 carbon atoms. In yet other embodiments, the two R⁵ groups combinewith the nitrogen atom to which they are attached to form a heterocyclicgroup. The heterocyclic group includes at least one nitrogen atom andcan contain other heteroatoms such as oxygen or sulfur. Exemplaryheterocyclic groups include, but are not limited to, imidazolyl,piperazinyl, and morpholinyl. The heterocyclic group can be fused to anadditional ring such as a benzene, cyclohexene, or cyclohexane.Exemplary heterocyclic groups fused to an additional ring include, butare not limited to, benzimidazolyl.

Exemplary amino (meth)acrylates of Formula (IIIb) where Q is oxyinclude, but are not limited to, N,N-dialkylaminoalkyl(meth)acrylatessuch as, for example, N,N-dimethylaminoethyl(meth)acrylate,N,N-diethylaminoethyl(meth)acrylate,N,N-dimethylaminopropyl(meth)acrylate,N-tert-butylaminopropyl(meth)acrylate, and the like.

Exemplary amino(meth)acrylamides of Formula (IIIb) where Q is —NH—include, but are not limited to, N-(3-aminopropyl)(meth)acrylamide,N-[3-(dimethylamino)propyl](meth)acrylamide,N-(3-imidazolylpropyl)(meth)acrylamide,N-(2-imidazolylethyl)(meth)acrylamide,N-(1,1-dimethyl-3-imidazoylpropyl)(meth)acrylamide, andN-(3-benzimidazolylpropyl)(meth)acrylamide.

Exemplary quaternary salts of the functional monomers of Formula (IIIb)include, but are not limited to, (meth)acrylamidoalkyltrimethylammoniumsalts such as (meth)acrylamidopropyltrimethylammonium chloride; and(meth)acryloxyalkyltrimethylammonium salts such as2-(meth)acryloxyethyltrimethylammonium chloride, and2-(meth)acryloxyethyltrimethylammonium methyl sulfate.

A third type of functional monomer of Formula (III) or (IIIa) has ahydroxyl Z¹ group. Suitable hydroxy-containing monomers include hydroxysubstituted alkyl(meth)acrylates, hydroxy substitutedalkyl(meth)acrylamides, or vinyl alcohols. Specific hydroxy-containingmonomers include, but are not limited to, 2-hydroxyethyl(meth)acrylate,3-hydroxylpropyl(meth)acrylate, 4-hydroxybutyl(meth)acrylate, glycerol(meth)acrylate, N-[tris(hydroxymethyl)methyl]acrylamide, vinylbenzylalcohol, and hydroxyethyl(meth)acrylamide.

A fourth type of functional monomer of Formula (III) or (IIIa) has anazlactone Z¹ group. Exemplary functional monomers having an azlactonegroup include, but are not limited to, vinyl alkylazlactones such as2-vinyl-4,4-dimethylazlactone (also called2-vinyl-4,4-dimethyl-2-oxazolin-5-one),2-(4-vinylphenyl)-4,4-dimethylazlactone,2-isopropenyl-4,4-dimethylazlactone,2-vinyl-4-ethyl-4-methyl-2-oxazolin-5-one, and2-vinyl-4,4-dimethyl-1,3-oxazin-6-one. A further embodiment of thefourth type of functional monomer has a precursor group of the azlactonegroup as the Z¹ group. The precursor group can be subjected to a ringclosure reaction to form the azlactone group. Exemplary functionalmonomers that can provide this precursor group include, but are notlimited to, N-acryloylmethylalanine.

A fifth type of functional monomer of Formula (III) or (IIIa) has aglycidyl as the Z¹ group. Exemplary monomers having a glycidyl groupinclude, but are not limited to, glycidyl(meth)acrylate.

Still other functional monomers have a combination of two or morefunctional Z¹ groups selected from (1) an acidic group or salt thereof,(2) an amino group or salt thereof, (3) a hydroxyl group, (4) anazlactone group, or (5) a glycidyl group. Exemplary functional monomershaving multiple and different types of functional groups are3-(meth)acryloxy-2-hydroxypropyltrimethylammonium chloride and2-(meth)acrylamidoglycolic acid.

The polymerization of the (meth)acryloyl-functionalized support ofFormula (II) with a monomer composition that contains a functionalmonomer of Formula (III) leads to the formation of the grafted supportof Formula (IV).SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²U²—CH₂—U¹  (IV)In Formula (IV), SS refers to the solid support. Groups Q, Y², Z¹, R¹,p, and Y¹ are the same as described for Formulas (II) and (III). GroupU¹ is a polymeric unit that includes at least one divalent monomericunit of formula —CR²(Y²Z¹)—CH₂—. Group U² is hydrogen or a group thatincludes at least one divalent monomeric unit of formula—CR²(Y²Z¹)—CH₂—. The Z¹ groups that are included in group U¹ or in bothgroups U¹ and U² can often react with or interact with a targetcompound.

The grafted chain can include one or more monomeric units of formula—CR²(Y²Z¹)—CH₂—. In many embodiments, the grafted chain includes atleast two or at least three monomeric units. That is, the grafted chainif often polymeric and includes at least two or at least three pendantgroups —Y²—Z¹.

The groups U¹ and U² can include at least one group of formula—CR²(Y²Z¹)—CH₂—. Often, these groups include at least two or at leastthree monomeric units of formula —CR²(Y²Z¹)—CH₂— and are considered tobe polymeric chains. In a few instances, the group U² or U¹ is a residueof a chain transfer agent used in the polymerization reaction or is aresidue of a free radical initiator molecule. If either U¹ or U² is theresidue of a chain transfer agent, it is often a halogen such as fromtetrabromomethane.

The grafted support can be used to separate and/or purify a targetcompound. That is, methods of separating or purifying a target compoundare provided that include providing a grafted support of Formula (IV)and contacting a sample containing the target compound with the graftedsupport. The target compound reacts with or interacts with thefunctional group Z¹ of the grafted chain of the grafted support.

In some embodiments of the grafted support of Formula (IV), the graftedchain has a Z¹ group that is an acidic group or a salt thereof. Thegrafted support can function as a cation exchange material. When the pHis suitably adjusted, the Z¹ group on the grafted chain can be anegatively charged group capable of interacting with a positivelycharged group of the target compound (i.e., the target compound is acation). The target compound can be adsorbed on the grafted support. Torelease the adsorbed target compound, the pH can be raised (e.g., the pHcan be raised to at least 6 or 7 or higher). Alternatively, when thetarget compound is a biomolecule, the sample can be contacted with andadsorbed on the grafted support in a low ionic strength buffer (e.g., 5to 150 millimolar (mM) buffer salt plus 0 to 200 millimolar sodiumchloride) at a pH of about 3 to 10 or at a pH of about 4 to 6. Torelease the adsorbed biomolecule, the grafted support can be contactedwith a high ionic strength buffer. In some embodiments, the high ionicstrength buffer includes that same buffer composition used to adsorb thetarget compound plus 1 or 2 molar (M) sodium chloride. The adsorptionand release processes are typically performed at temperatures near roomtemperature.

The grafted supports of Formula (IV) can often be used under pHconditions and/or salt conditions that may be unsuitable for some knownion exchange resins. For example, the dynamic binding capacity of thegrafted support can have a maximum at a pH that is 0.5 or 1 pH unithigher or lower than many known ion exchange resins. Greater capacity ata higher pH value can be particularly advantageous for the separation orpurification of various proteins. Many proteins are sensitive to low pHconditions and many known ion exchange resins tend to be used at pHvalues that are not optimal for proteins. The grafted supports can beused at higher pH values that are more suitable for some proteins.

Buffer salts useful for controlling the pH for cation exchange reactionsinclude, but are not limited to, sodium phosphate, sodium carbonate,sodium bicarbonate, sodium borate, sodium acetate, and TRIS(tris(hydroxymethyl)aminomethane). Other suitable buffers include“Good's” buffers such as MOPS (3-morpholinopropanesulfonic acid), EPPS(4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), MES(2-morpholinoethanesulfonic acid), and others.

In other embodiments of the grafted support of Formula (IV), the graftedchain has a Z¹ group that is an amino group or a salt thereof. A primaryamino group or a secondary amino group can react as a nucleophilic agentwith a target compound. Alternatively, the grafted support can functionas an anion exchange material. When the pH is suitably adjusted, thegrafted support can have a positively charged group capable ofinteracting with a negatively charged group of the target compound(i.e., the target compound is an anion).

In general, in order to get effective adsorption of the negativelycharged target compound to the anion exchange material, a pH of at leastabout 1 to 2 pH units above the pKa of the target compound (or pI for aprotein) can be used. To release the adsorbed target compound from theanion exchange material, if desired, the pH can be lowered at least 1 to2 pH units, or more. Alternatively, when the charged target compound isa biomolecule, the sample can be contacted with the anion exchangematerial in a low ionic strength buffer (e.g., a 5 to 20 millimolarbuffer salt) at an appropriate pH (e.g., at a pH of about 6-8 for bovineserum albumin). To release the adsorbed biomolecule, the anionicexchange material is often contacted with a high ionic strength buffer.In some embodiments, the high ionic strength buffer includes that samebuffer composition used to adsorb the target compound plus 1 molarsodium chloride. The adsorption and release processes are typicallyperformed at temperatures near room temperature.

Buffer salts useful for controlling pH for anion exchange materialsinclude, but are not limited to, sodium phosphate, sodium carbonate,sodium bicarbonate, sodium borate, sodium acetate, and TRIS(tris(hydroxymethyl)aminomethane). Other suitable buffers include“Good's” buffers such as MOPS (3-morpholinopropanesulfonic acid), EPPS(4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), MES(2-morpholinoethanesulfonic acid), and others.

In still other embodiments of the grafted support of Formula (IV), thegrafted chain has a hydroxyl Z¹ group. The hydroxyl group on the graftedchain can undergo a condensation reaction with a target compound. Forexample, a hydroxyl group can react with a target compound having acarboxyl group (—COOH) to form an ester. That is, the reaction resultsin the formation of a carbonyloxy linkage group that covalently bondsthe target compound to the grafted chain. For example, a protein orother molecule can be covalently bonded to the grafted chain.Alternatively, the grafted chains having hydroxyl groups can serve toprovide a neutral, hydrophilic, pore-modifying material such that theresin can be used as a size exclusion resin.

In yet other embodiments of the grafted support of Formula (IV), thegrafted chain has an azlactone Z¹ group. The azlactone group on thegrafted chain can undergo a ring-opening reaction with a target compoundhaving a nucleophilic group. Suitable nucleophilic groups for reactingwith an azlactone group include, but are not limited to, primary aminogroups, secondary amino groups, thiol groups, and hydroxyl groups. Thereaction of the azlactone group with a nucleophilic group of the targetcompound usually results in the formation of a linkage group thatattaches the target compound to the grafted chain. The linkage groupformed by ring-opening of the azlactone group often contains the group—(CO)NHC(R¹)₂(CH₂)_(p)(CO)—. The reaction of azlactone-functional resinswith a variety of nucleophilic compounds (e.g., target compounds) isfurther described in U.S. Pat. No. 5,292,840 (Heilmann et al.), U.S.Pat. No. 5,561,097 (Gleason et al.), and U.S. Pat. No. 6,379,952(Rasmussen et al.).

Alternatively, the grafted support of Formula (IV) can have a graftedchain that includes a precursor group of the azlactone group. Theseprecursor group can be subjected to a ring closure reaction to form theazlactone group. Once formed, the azlactone group can react as describedabove with various target compounds. The ring closure reaction canoccur, for example, by treating the grafted support of Formula (IV) withacetic anhydride, trifluoroacetic anhydride, or alkyl chloroformates.This process is further described in U.S. Pat. No. 5,403,902 (Heilmannet al.) and U.S. Pat. No. 5,336,742 (Heilmann et al.).

In further embodiments of the grafted support of Formula (IV), thegrafted chain has a glycidyl Z¹ group. The glycidyl group can undergo aring-opening reaction with a target compound having a nucleophilicgroup. Suitable nucleophilic groups for reacting with a glycidyl groupinclude, but are not limited to, primary amino groups, secondary aminogroups, hydroxyl groups, tertiary amino groups, thiol groups, andcarboxyl groups. The reaction of the glycidyl group with a nucleophilicgroup of the target compound usually results in the formation of alinkage group that functions to attach the target compound to thegrafted chains. The linkage group formed by ring-opening of the glycidylgroup often contains the group —C(OH)HCH₂—. The linkage group can be,for example, —C(OH)HCH₂NR³— when the glycidyl group is reacted with anamino group, —C(OH)HCH₂O— when the glycidyl group is reacted with ahydroxyl group, —C(OH)HCH₂S— when the glycidyl group is reacted with athiol group, or —C(OH)HCH₂O(CO)— when the glycidyl group is reacted witha carboxyl group.

In some applications, the grafted support of Formula (IV) can be furthermodified to provide other groups for interaction with or reaction with atarget compound. In many embodiments, a modifying agent of formula A-Tis reacted with the grafted support. In the formula A-T, group A is themodifying group and T is a remainder of the modifying agent and is equalto the modifying agent A-T minus the modifying group A. The modifyinggroup A reacts with the functional group Z¹ on the grafted chains. Thereaction of the functional group Z¹ and the modifying group A results inthe formation of an attachment group L. The attachment group L isattached to T, which is the remainder of the modifying agent. Thereaction is shown in Reaction Scheme C. G-Z¹ is used to refer to thegrafted support of Formula (IV) that has a functional group Z¹ on thegrafted chain.

Reaction Scheme C

G-Z¹+A-T→G-L-TThe formula G-Z¹ contains only one Z¹ group for ease of discussion. Manygrafted supports have multiple grafted chains and many of the graftedchains have multiple Z¹ groups. Exemplary modifying agents are shown inTable 1 for each type of Z¹ functional group. In Table 1, the group Xrefers to a halo (e.g., X⁻ is a halide) and group D is selected fromoxy, thio, or —NR³—. Each group R⁴ is independently selected fromhydrogen, alkyl, heteroalkyl, aryl, or aralkyl. Group R⁵ is an alkylene.The group Az refers to an azlactone group and the group Az¹ refers to aring-opened azlactone group.

TABLE 1 Reaction of Grafted Support with Modifying Agent Group G-Z¹Modifying Agent A—T G—L—T G—(CO)OH HD—T G—(CO)D—T

G—(CO)O—C(R⁴)₂—C(R⁴)₂—NH(CO)—T

G—(CO)O—C(R⁴)₂—C(R⁴)₂—NH—T

G—(CO)O—C(R⁴)₂—C(R⁴)(DH)—T or G—(CO)O—C(R⁴)(T)—C(R⁴)₂(DH) O═C═N—TG—(CO)—NH—T G—OH O═C═N—T G—O(CO)—NH—T X—(CO)—T G—O(CO)—T HO—(CO)—TG—O(CO)—T

G—O—C(R⁴)₂—C(R⁴)₂—NH—T

G—O—C(R⁴)₂—C(R⁴)(DH)—T or G—O—C(R⁴)(T)—C(R⁴)₂(DH)

G—O(CO)—C(R⁴)2—NH—(CO)—T

G—O(CO)—T G—N(R³)H O═C═N—T G—N(R³)(CO)—NH—T X—(CO)—T G—N(R³)(CO)—THO—(CO)—T G—N(R³)(CO)—T

G—N(R³)—C(R⁴)₂—C(R⁴)₂—NH—T

G—N(R³)—C(R⁴)₂—C(R⁴)(DH)(T) or G—N(R³)—C(R⁴)(T)—C(R⁴)₂(DH)

G—N(R³)(CO)—C(R4)2—NH—(CO)—T X—R⁵—T G—NR³—R⁵—T

G—N(R³)(CO)—T G—N(R³)₂ X—R⁵—T G—N⁺(R³)₂—R⁵—T X⁻ G—Az HD—T G—Az¹—D—T

HD—T G—C(OH)R⁴—C(R⁴)₂(D—T) or G—C(D-T)R⁴—C(R⁴)₂(OH) T—(CO)OHG—C(OH)(R⁴)—C(R⁴)₂—OOC—T or G—C(R⁴)(OOC—T)—C(R⁴)₂(OH)

The modified solid support can be represented by Formula (V).SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²U⁴—CH₂—U³  (V)In Formula (V), SS is a solid support and R¹ is each independentlyselected from alkyl, heteroalkyl, aryl, or aralkyl. The group R² ishydrogen or an alkyl. The variable p is an integer equal to 0 or 1. Eachgroup Q is independently a divalent group selected from oxy, thio, or—NR³— where R³ is hydrogen, alkyl, heteroalkyl, aryl, or aralkyl. Thegroup Y¹ is a first linking group containing an alkylene,heteroalkylene, arylene, or combination thereof. Y¹ optionally caninclude an oxy, thio, amino, carbonylimino, carbonyloxy, carbonylthio,or a combination thereof separating one or more alkylenes,heteroalkylenes, arylenes, or mixtures thereof. The group U³ includes atleast one divalent monomeric unit of formula —CR²(Y²-L-T)-CH₂—. Group U⁴is selected from hydrogen or a group that includes at least one divalentmonomeric unit of formula —CR²(Y²-L-T)-CH₂—. Group L is an attachmentgroup formed by reacting a group Z¹ with a modifying group of amodifying agent of formula A-T where A is the modifying group. Group Tis a remainder of the modifying agent A-T and is equal to the modifyingagent A-T minus the modifying group A. Group Z¹ is a functional groupselected from (1) an acidic group or a salt thereof, (2) an amino groupor a salt thereof, (3) a hydroxyl group, (4) an azlactone group or aprecursor of the azlactone group, (5) a glycidyl group, or (6) acombination thereof.

The modified grafted support can be used to separate and/or purify atarget compound. That is, methods of separating or purifying a targetcompound are provided that include providing a grafted support ofFormula (V) and contacting a sample containing the target compound withthe grafted support. The target compound reacts with or interacts withthe remainder group T of the modified grafted support. The modifiedgrafted supports can function as affinity resins or materials, ionexchange resins or materials, hydrophobic interaction resins ormaterials, reverse phase resins or materials, size exclusion resins ormaterials, chelating resins or materials, cell selection resins ormaterials, immobilized enzyme resins or materials, mixed mode resins ormaterials, or the like.

Affinity resins or materials can be prepared, for example, by reacting agroup Z¹ with a modifying agent that includes a modifying group plus anaffinity ligand. An affinity ligand is a group or compound that can bindanother group or compound. For example an azlactone group or a glycidylZ¹ group on the grafted support of Formula (IV) can react with amodifying agent that contains a nucleophilic modifying group plus anaffinity ligand. More particularly, an amino group of a biomolecule canreact with the azlactone or glycidyl group to covalently attach thebiomolecule to the grafted support. The attached biomolecule caninteract with a complementary biomolecule. Exemplary modifying agentsinclude an antigen that can bind to a corresponding (i.e.,complementary) antibody or an antibody that can bind to a corresponding(i.e., complementary) antigen. Other exemplary modifying agents includea DNA or RNA fragment that can bind with a complementary DNA or RNAfragment and a lectin that can bind with a compound or biomoleculecontaining a carbohydrate moiety.

Ion exchange resins or materials can be prepared, for example, byreacting Z¹ on the grafted support of Formula (IV) with a modifyingagent having a modifying group plus an ionic group. For example, themodifying agent can have a nucleophilic modifying group plus a secondgroup that is basic, acidic, or a salt thereof. The nucleophilic groupcan react with an azlactone or glycidyl Z¹ group resulting in theattachment of an ionic group (i.e., acidic group, basic group, or saltthereof) to the grafted support. Suitable modifying agents having both anucleophilic group and an ionic group include, but are not limited to,2-aminoethylsulfonic acid or aminopropyldimethylamine.

Hydrophobic interaction resins or materials can be prepared, forexample, by reacting a group Z¹ on the grafted support of Formula (IV)with a modifying agent having modifying group plus a hydrophobic group.For example, the modifying agent can have a nucleophilic modifying groupthat can react with an azlactone group or glycidyl Z¹ group resulting inthe attachment of the hydrophobic group to the grafted support. Suitablemodifying agents having both a nucleophilic group and a hydrophobicgroup include, but are not limited to, benzylamine, butylamine,hexylamine, or phenethylamine. Hydrophobic interaction resins can beused, for example, for purifying or separating relatively largemolecules such as proteins.

Reverse phase resins or materials can be prepared, for example, usingsimilar modifying agents to those used to prepare hydrophobicinteraction resins. That is, reverse phase resins can be prepared byreacting an azlactone group or a glycidyl Z¹ group on a grafted supportof Formula (IV) with a modifying agent having a nucleophilic modifyinggroup and a second group that is hydrophobic. The nucleophilic group canreact with the azlactone or the glycidyl Z¹ group resulting in theattachment of the modifying agent having a hydrophobic group to thegrafted support. Suitable modifying agents having a nucleophilic groupand a hydrophobic group include, for example, octyldecylamine. Withreverse phase interaction resins, the eluent is usually an organicsolvent rather than an aqueous-based solution. Further, reverse phaseresins are typically used for the separation or purification ofrelatively small molecules and peptides rather than proteins.

Size exclusion resins or materials can be prepared, for example, byreacting a group Z¹ on the grafted support of Formula (IV) with amodifying agent having a modifying group and a second group that isnon-interactive or neutral. For example, the modifying agent can includea nucleophilic modifying group that can react with an azlactone orglycidyl Z¹ group resulting in the attachment of the non-interactive orneutral group. Suitable modifying agents having both a nucleophilicgroup and a non-interactive or neutral group that can react with anazlactone Z¹ group include various amines, mercaptans, alcohols, oralcohol amines. For example, the modifying agent can be ethanolamine,ethanol, or ethylamine. Suitable modifying agents having both anucleophilic group and a non-interactive or neutral group that can reactwith a glycidyl Z¹ group include various carboxylic acids and alcohols.For example, the modifying agent can be ethanol or acetic acid.

Chelating resins or materials can be prepared, for example, by reactingZ¹ on the grafted support Formula (IV) with a modifying agent havingboth a modifying group and a second group that is metal-chelating. Forexample, the modifying agent can have a nucleophilic modifying groupthat can react with an azlactone or glycidyl Z¹ group resulting in theattachment of the metal-chelating group. Suitable modifying agentsinclude, but are not limited to, iminodiacetic acid,N-(3-aminopropyl)iminodiacetic acid, and N-(2-hydroxyethyl)iminodiaceticacid. The metal-chelating group, after chelation of a metal ion, caninteract, for example, with certain groups on proteins such as histidinegroups.

Cell selection resins can be prepared, for example, by reacting Z¹ onthe grafted support of Formula (IV) with an antibody to a cell surfacemarker. That is, the antibody is the modifying agent. The antibodytypically has a nucleophilic group such as an amino group that can reactwith an azlactone group or glycidyl group to attach the antibody to thegrafted support. The antibody can in turn bind with a cell surfacemarker on the cell resulting in the attachment of the cell to thegrafted support. Cell selection resins can be used, for example, topurify or separate stem cells, blood cells, or bacteria.

Immobilized enzyme resins can be prepared by reacting an azlactone or aglycidyl Z¹ group on the polymeric resin with a nucleophilic group of anenzyme to attach the enzyme to the grafted support. For example, theenzyme can be Penicillin G-acylase or glucoamylase. Immobilized enzymeresins can be used as catalysts for various reactions.

Mixed mode resins can be prepared by reacting a Z¹ group on the graftedsupport of Formula (IV) with modifying agents having a modifying groupplus additional groups that can impart two or more interaction modes tothe grafted support. The two or more modes of interaction can be any ofthose mentioned above. For example, an azlactone group or glycidyl groupcan be reacted with a modifying agent such as phenylalanine where theamino group would function as the nucleophilic group, the phenyl groupwould function as a hydrophobic group, and the carboxyl group wouldfunction as an ionic group.

In some embodiments, the grafted support or the modified grafted supportis placed within a chromatographic column. The chromatographic columncan be part of an analytical instrument or can be part of a preparativesystem. The preparative system can be of any suitable scale such as alaboratory scale, pilot plant scale, or industrial scale. In otherembodiments, the grafted support or the modified grafted support can bedisposed on a surface of a filtration medium. Any suitable filtrationmedium can be used. The filtration medium can be positioned within acartridge to provide a filter cartridge. In many applications, thegrafted supports or modified grafted supports are in the form of beads.The beads can have any suitable size.

EXAMPLES

These examples are merely for illustrative purposes and are not meant tobe limiting on the scope of the appended claims. All parts, percentages,ratios, and the like in the examples and the rest of the specificationare by weight, unless noted otherwise. Solvents and other reagents usedwere obtained from Sigma-Aldrich Chemical Company (Milwaukee, Wis.)unless otherwise noted.

Glossary of Terms EMPHAZE Polymeric beads commercially available fromAB1 3M Company (Saint Paul, MN) that are a reaction product ofmethylenebisacrylamide and 2-vinyl-4,4-dimethylazlac- tone. The beadscontain about 5 weight percent azlactone. The average size is typicallyabout 50 to 100 micrometers. MBA Methylenebisacrylamide VDM2-vinyl-4,4-dimethylazlactone, available from SNPE, Inc, Princeton, NJHEMA 2-Hydroxyethylmethacrylate HEA 2-Hydroxyethylacrylate PolyethylenePolyethylene glycol having a weight average molecular glycol 1000 weightof about 1000 g/mol TMEDA Tetramethylethylenediamine DMSODimethylsulfoxide Triton X-100 Nonionic surfactant APTACAcrylamidopropyl trimethylammonium chloride MAPTAC(3-methacrylamidopropyl)trimethylammonium chloride MOPS3-(N-morpholino)propanesulfonic acid IgG Human polyclonal ImmunoglobulinG available from Equitech-Bio, Kerrville, TX BSA Bovine Serum AlbuminProtein A Stock aqueous solution, approximately 50 mg/mL, available fromRepligen Corporation, Waltham, MATest MethodsStatic Cation Exchange Capacity for Immunoglobulin G (IgG)

A 50 volume percent slurry of cation exchange polymeric beads wasprepared by mixing the polymeric beads with deionized water,centrifuging at 3000 relative centrifugal force (rcf) for 20 minutes toform a packed bead bed, and then adjusting the amount of deionized waterso that the total volume was twice that of the packed bead bed. Theslurry was mixed well to suspend the polymeric beads, and then a 400microliter sample of the slurry was pipetted into a 5 mL, 0.45micrometer cellulose acetate centrifugal microfilter that iscommercially available under the trade designation CENTREX MF throughVWR (Eagan, Minn.). The water was removed by centrifugation at 3000 rcffor 5 minutes. The polymeric beads were then mixed with 4 mL of a buffercontaining 50 mM sodium acetate and 80 mM sodium chloride at pH 4.5. Thesample was centrifuged again at 3000 rcf for 10 minutes. The supernatewas discarded. Then a 4.5 mL sample of IgG, having a concentration ofabout 7 mg/mL in the same acetate buffer was added to the filtercontaining the polymeric beads. The mixture was mixed by tumblingovernight, and then the supernate was removed from the polymeric beadsby centrifugation at 3000 rcf for 20 min.

The supernate was analyzed by UV spectroscopy. The absorbance of thesample at 280 nm was compared to that of the starting IgG solution. Thedifference was used to calculate the IgG capacity of the polymericbeads. Assays were run in triplicate and averaged.

Cationic Dynamic Binding Capacity (DBC) for Immunoglobulin G (IgG)

An aqueous slurry of polymeric beads (approximately 350 microliter totalvolume of polymeric beads) was packed into a 5 centimeters by 0.3centimeter inner diameter glass column commercially available under thetrade designation OMNIFIT from Chromtech (Apple Valley, Minn.), placedon a Fast Protein Liquid Chromatograph commercially available under thetrade designation AKTA from GE Healthcare (Uppsala, Sweden), andequilibrated for 10 column volumes with Buffer A (50 mM acetate, 40 mMNaCl) at 0.7 mL/minute. The pH of Buffer A was 4.5 unless otherwisenoted otherwise. The challenge solution (5.0 mg/mL human IgG in bufferA) was loaded at 0.09 mL/min (3.9 minutes residence time/76 cm/hr) until7 mL of sample was loaded or the UV absorbance at a wavelength of 280nanometers (A₂₈₀) exceeded 800 mAU (whichever came first). The amount ofIgG bound to the support was determined at the point where theconcentration of the solution exiting the column during the initialloading was 10 percent of the initial IgG challenge solutionconcentration (the plateau of non-binding proteins was subtracted out).

Small Ion Capacity (SIC) for Hydrogen Ion

Approximately 8 mL of a polymeric bead slurry (approximately 50 volumepercent in deionized water) was transferred to a 15 mL graduatedcentrifuge tube and centrifuged at 3000 relative centrifugal force (rcf)for 5 minutes. The volume of the resulting packed polymeric beads wasrecorded to the nearest 0.1 mL and the slurry was transferredquantitatively to a sintered glass funnel and washed with deionizedwater (3×50 mL), with 0.5N HCl (3×50 mL), and then again with deionizedwater (3×50 mL). The washed polymeric beads were then quantitativelytransferred to a 125 mL Erlenmeyer flask and 4.0 mL of 2M NaCl was addedto displace the hydrogen ions. After 5 minutes, 2 drops ofphenolphthalein solution (1 gram in 100 mL ethanol) were added to theslurry and the mixture was titrated (while mixing on a magnetic stirplate) with 0.1 N NaOH until the solution was faint pink. The small ioncapacity in micromoles per mL of polymeric beads was calculated bydividing the volume of 0.1 NaOH added by the volume of beads analyzedand multiplying by 100.

Static Anion Exchange Capacity for Bovine Serum Albumin (BSA)

A 50 volume percent slurry of anion exchange polymeric beads wasprepared by mixing the polymeric beads with deionized water,centrifuging at 3000 relative centrifugal force (rcf) for 20 minutes toform a packed bead bed, and then adjusting the amount of deionized waterso that the total volume was twice that of the packed bead bed. Theslurry was mixed well to suspend the polymeric beads, and then a 400microliter sample of the slurry was pipetted into a 5 mL, 0.45micrometer cellulose acetate centrifugal microfilter that iscommercially available under the trade designation CENTREX MF throughVWR (Eagan, Minn.). The water was removed by centrifugation at 3000 rcffor 5 minutes. The polymeric beads were then mixed with 4 mL of a buffercontaining 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.5.The sample was centrifuged again at 3000 rcf for 10 minutes. Thesupernate was discarded. Then a 4.5 mL sample of BSA, which was obtainedfrom Sigma-Aldrich (St. Louis, Mo.), having a concentration of about 9mg/mL in the same MOPS buffer was added to the filter containing thepolymeric beads. The mixture was mixed by tumbling overnight, and thenthe supernate was removed from the polymeric beads by centrifugation at3000 rcf for 20 min.

The supernate was analyzed by UV spectroscopy. The absorbance of thesample at 279 nm was compared to that of the starting BSA solution. Thedifference was used to calculate the BSA capacity of the polymericbeads. Assays were run in triplicate and averaged.

Anionic Dynamic Binding Capacity (DBC) for BSA

An aqueous slurry of polymeric beads (approximately 350 microliter totalvolume of polymeric beads) was packed into a 5 centimeters by 0.3centimeter inner diameter glass column commercially available under thetrade designation OMNIFIT from Chromtech (Apple Valley, Minn.), placedon a Fast Protein Liquid Chromatograph commercially available under thetrade designation AKTA from GE Healthcare (Uppsala, Sweden), andequilibrated for 9 column volumes with Buffer A (25 mMtris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 8.0) at 0.5mL/minute. The challenge solution (5.0 mg/mL BSA in buffer A) was loadedat 0.1 mL/min (3.5 minutes residence time/85 cm/hr) until 15 mL ofsample was loaded or the UV absorbance at a wavelength of 280 nanometers(A₂₈₀) exceeded 250 mAU (whichever came first). The amount of BSA boundto the support was determined at the point where the concentration ofthe solution exiting the column during the initial loading was 10percent of the initial BSA challenge solution concentration.

Small Ion Capacity (SIC) for Hydroxide Ion

Approximately 8 mL of a polymeric bead slurry (approximately 50 volumepercent in deionized water) was transferred to a 15 mL graduatedcentrifuge tube and centrifuged at 3000 relative centrifugal force (rcf)for 5 minutes. The volume of the resulting packed polymeric beads wasrecorded to the nearest 0.1 mL and the slurry was transferredquantitatively to a sintered glass funnel and washed with deionizedwater (3×50 mL), with 0.1N NaOH (3×50 mL), and then again with deionizedwater (3×50 mL). The washed polymeric beads were then quantitativelytransferred to a 125 mL Erlenmeyer flask and 4.0 mL of 2M sodium sulfatewas added to displace the hydroxide ions. After 5 minutes, 2 drops ofphenolphthalein solution (1 gram in 100 mL ethanol) were added to theslurry and the mixture was titrated (while mixing on a magnetic stirplate) with 0.1 N HCl until the color of the solution changed from pinkto colorless. The small ion capacity in micromoles per mL of polymericbeads was calculated by dividing the volume of 0.1 HCl added by thevolume of beads analyzed and multiplying by 100.

Static Affinity Binding Capacity for Immunoglobulin G (IgG)

A 50 volume percent slurry of Protein A derivatized polymeric beads wasprepared by mixing the polymeric beads with deionized water,centrifuging at 3000 relative centrifugal force (rcf) for 20 minutes toform a packed bead bed, and then adjusting the amount of deionized waterso that the total volume was twice that of the packed bead bed. Theslurry was mixed well to suspend the polymeric beads, and then a 200microliter sample of the slurry was pipetted into a 5 mL, 0.45micrometer cellulose acetate centrifugal microfilter that iscommercially available under the trade designation CENTREX MF throughVWR (Eagan, Minn.). The water was removed by centrifugation at 3000 rcffor 5 minutes. The polymeric beads were then mixed with 2.25 mL of ahuman IgG solution (about 5 mg/mL hIgG, in 10 mM phosphate, 150 mMsodium chloride, pH 7.4). The mixture was mixed by tumbling overnight,and then the supernate was removed from the polymeric beads bycentrifugation at 3000 rcf for 5 min.

The supernate was analyzed by UV spectroscopy. The absorbance of thesample at 280 nm was compared to that of the starting IgG solution. Thedifference was used to calculate the IgG capacity of the polymericbeads. Assays were run in triplicate and averaged.

Affinity Dynamic Binding Capacity (DBC) for Immunoglobulin G (IgG)

An aqueous slurry of Protein A derivatized polymeric beads(approximately 350 microliter total volume of polymeric beads) waspacked into a 5 centimeters by 0.3 centimeter inner diameter glasscolumn commercially available under the trade designation OMNIFIT fromChromtech (Apple Valley, Minn.), placed on a Fast Protein LiquidChromatograph commercially available under the trade designation AKTAfrom GE Healthcare (Uppsala, Sweden), and equilibrated for 10 columnvolumes with Buffer A (10 mM phosphate, 150 mM sodium chloride, pH 7.4,spiked with 0.01% w/v sodium azide) at 0.7 mL/minute. The challengesolution (3.0 mg/mL human IgG in buffer A) was loaded at 0.09 mL/min(3.9 minutes residence time/76 cm/hr) until 7 mL of sample was loaded orthe UV absorbance at a wavelength of 280 nanometers (A₂₈₀) exceeded 300mAU (whichever came first). A washout of unbound sample was performed byflowing Buffer A at 0.7 mL/min flow rate for 18 column volumes. This wasfollowed by isocratic elution with Buffer B (2% v/v glacial acetic acid,0.1 M glycine) for 9 column volumes. The amount of IgG bound to thesupport was determined at the point where the concentration of thesolution exiting the column during the initial loading was 10 percent ofthe initial IgG challenge solution concentration (the plateau ofnon-binding proteins was subtracted out). The column was thenre-equilibrated by flowing 15 column volumes of Buffer A.

Example 1

A 15 gram sample of EMPHAZE AB1 beads was slurried in ethyl acetate (250mL) in a 1 L round bottomed flask with overhead stirrer.2-Hydroxyethylmethacrylate (HEMA, 15 mL) was added. After stirring for 5minutes, borontrifluoride diethyletherate (300 μL) was added, and themixture was allowed to react for 72 hours at ambient temperature. Thebead slurry was then filtered, washed with acetone (4×250 mL), and driedovernight under vacuum.

Example 2

Example 1 was repeated except that 2-hydroxyethylacrylate (HEA) was usedin place of HEMA.

Example 3

A 2 L split resin flask (Morton type) equipped with an overhead stirrer,heating control, reflux condenser, and nitrogen inlet was charged withtoluene (188 mL) and polymeric stabilizer (0.13 g). The polymericstabilizer was a 91.8:8.2 by weight copolymer of isooctylacrylate and2-acrylamidoisobutyramide that was prepared as described in Rasmussen,et al., Makromol. Chem., Macromol. Symp., 54/55, 535-550 (1992). Thesolution was stirred at 450 rpm until all the stabilizer had dissolved.Heptane (348 mL) was added, and the mixture was heated under a slownitrogen purge until the temperature equilibrated to 35° C. MBA (13.86grams), and 2-acrylamido-2-methylalanine (0.14 gram) were weighed into a225 mL Erlenmeyer flask. To the flask were added isopropanol (80 mL),deionized (DI) water (38.3 mL), 1N sodium hydroxide (1.78 mL), andpolyethylene glycol 1000 (20 mL of a 50% w/w solution in DI water). Themixture was stirred at low heat until all monomers had dissolved. Asolution of sodium persulfate (0.56 grams) in DI water (5 mL) was addedwith swirling to the monomer solution. The resulting solution wasimmediately added to the equilibrated reaction flask. Stirring andpurging was continued until the batch had re-equilibrated to 35° C. Thentetramethylethylenediamine (TMEDA, 0.56 mL) was added to initiatepolymerization. The polymerization reaction was allowed to proceed for atotal of two hours. The bead suspension was filtered and washed withacetone (2×500 mL), methanol (2×500 mL), and again with acetone (2×500mL). The wet filter cake was transferred to an Erlenmeyer flask,suspended in acetone (250 mL), and sonicated with swirling for 10minutes to break up agglomerates. The beads were filtered, reslurried inwater, and sieved using a RO-TAP sieve (W.S. Tyler, Mentor Ohio). Thesize fraction between 38-90 micrometers in diameter was collected. Themean particle size was determined to be 82.3 micrometers.

Approximately 20 mL of hydrated bead slurry was filtered and then washedwith 0.1 N hydrochloric acid (2×50 mL), DI water (2×50 mL), acetone(3×50 ml), and dimethylsulfoxide (DMSO, 3×50 mL). The damp beads wereplaced in a 50 mL polypropylene centrifuge tube and diluted up to twicethe swollen volume with DMSO. Acetic anhydride (1.7 mL) andtriethylamine (0.1 mL) were added and the mixture was mixed for 4 hoursat 25° C. The beads were filtered, washed with acetone (3×50 mL), anddried overnight under high vacuum. The dried beads were then reactedwith HEMA as described in Example 1.

Example 4

Ethylenediamine (12.0 grams) was dissolved in isopropanol (200 mL), thena 20 gram sample of EMPHAZE AB1 beads was added. The slurry was mixedfor 2 hours at ambient temperature, filtered, and then washed withisopropanol (3×200 mL), distilled water (4×200 mL), 0.1 N HCl (3×200ml), and distilled water (5×200 mL). The resultant amine-functionalbeads, when titrated by the procedure above for small ion exchangecapacity for hydrogen ion, were determined to have an aminefunctionality of about 29 micromoles/mL of bead.

Half of the above slurry was filtered, washed with isopropanol (100 mL),isopropanol (100 mL) containing 0.1 N NaOH (20 mL) and isopropanol(3×100 mL). The wet cake was then slurried in isopropanol (about 50% byvolume slurry), VDM (5 mL) was added, and the mixture was allowed to mixfor 2 hours at ambient temperature. The beads were then filtered, washedwith acetone (3×100 mL) and dried on a rotary evaporator at 60° C. fortwo hours to provide about 10 grams of acrylamide-functional beads,suitable for grafting reactions.

Example 5

Toluene (100 mL), heptane (300 mL), and Triton X-100 (1000 μL) wereadded to a 3-necked round bottomed flask. The mixture was purged withnitrogen while stirring with an overhead stirrer at room temperature.

In a separate 125 mL Erlenmeyer flask was dissolved AMPS (30 grams of a50% w/w solution in water), sodium persulfate (0.20 grams), isopropanol(35 mL), and water (20 mL). HEMA-functionalized beads from Example 3(2.0 grams) were added to the aqueous mixture and allowed to soak for 10minutes. The beads were filtered to remove excess liquid and thentransferred into the organic toluene/heptane mixture. The suspendedbeads were stirred and purged with nitrogen for 30 minutes before addingtetramethylethylenediamine (TMEDA, 0.2 mL). The mixture was stirred for1 hour. The beads were filtered, washed with both acetone (3×100 mL) andDI water (3×100 mL), and then stored as a slurry in DI water for furtheranalysis. The analysis of this material is shown in Table 1.

Examples 6 to 9

The procedure used to prepare Example 5 was repeated but with varyingamounts of AMPS solution (10, 15, 20, and 40 grams, respectively). Theamount of DI water was adjusted so that each reaction had a total of 35mL of water. The analyses of these materials are shown in Table 1.

Examples 10 to 13

Examples 6 to 9 were repeated using the HEMA-functional beads ofPreparative Example 1 along with 5, 10, 20, and 30 grams of AMPSsolution, respectively. The analyses of these materials are shown inTable 1.

Examples 14 to 18

Example 12 was repeated, except that the amounts of isopropanol andwater were varied. The amounts used were 0/70, 20/50, 49/21, and 60/10mL, respectively, for Examples 14 to 17. For Example 18, 7.5 grams ofAMPS solution, 7.5 mL isopropanol, and 18.75 mL DI water were used. Theanalyses of these materials are shown in Table 1.

Comparative Example 1

A 98:2 w/w MBA/AMA bead was prepared by a procedure similar to that ofExample 3. This bead, without derivatization with HEMA, was subjected tothe grafting procedure of Example 18. Small ion capacity of theresultant bead was 31 μmol/mL. This low small ion capacity indicatesthat very little, if any, grafting occurred.

TABLE 1 Characterization of Cation Exchange Beads SIC Static IgG DBC DBCDBC Example (μmol/mL) (mg/mL) (pH 4.5) (pH 5.0) (pH 5.5) 5 202 114 15.157.2 20.7 6 36 87 42.6 59.1 26.9 7 100 150 20.3 79.5 62.9 8 99 96 28.171.3 29.5 9 180 79 18.0 55.2 24.5 10 88 82 42.5 11 149 115 56.5 12 253151 55.9 13 314 107 49.4 14 251 9 17.6 15 236 130 40.1 16 261 113 37.917 330 102 28.3 18 193 126 78.8

Example 19

The grafting procedure of Example 5 was repeated using HEMA-functionalbeads from Example 1 (2.0 grams), substituting APTAC (20 grams of a 75%solution by weight in water) for AMPS. The appropriate adjustment wasmade for the amount of added DI water due to the fact that the APTACmonomer is a 75% by weight solution in water rather than a 50% by weightsolution in water. Characterization of the resultant grafted beads isshown in Table 2.

Examples 20-24

Example 19 was repeated, except that the amount of APTAC solution usedwas 15, 10, 7.5, 5, and 2.5 grams respectively. The amounts of added DIwater were adjusted to provide a total of 17.5 mL water.Characterization of the resultant grafted beads is shown in Table 2.

Examples 25-29

Example 19 was repeated, using HEMA-functional beads from Example 1 (1gram), and substituting varying amounts of(3-methacrylamidopropyl)trimethylammonium chloride (MAPTAC, 50% solutionby weight in water) for APTAC. Again, adjustment were made to theamounts of DI water added to provide a total of 17.5 mL water. Amountsof MAPTAC solution used were 20, 15, 10, 7.5, and 5 grams, respectively.Characterization of the resultant grafted beads is shown in Table 2.

TABLE 2 Grafted Anion Exchange Beads SIC Static BSA DBC Example(μmol/mL) (mg/mL) (pH 8.0) 19 442 75.3 45.9 20 382 99.7 69.2 21 300143.5 92.6 22 188 169.0 89.2 23 123 161.9 74.7 24 55 89.4 50.0 25 260144.3 81.2 26 192 132.2 95.5 27 161 138.2 82.6 28 133 137.0 82.2 29 95116.5 49.6

Example 30

Toluene (50 mL), heptane (150 mL), and Triton X-100 (500 μL) were addedto a 3-necked round bottomed flask. The mixture was purged with nitrogenwhile stirring with an overhead stirrer at room temperature.

N-acryloyl-2-methylalanine (AMA, 30 milligrams), methacrylamide (MA, 544milligrams), sodium persulfate (0.12 grams), isopropanol (17.5 mL), 0.1N sodium hydroxide (1.92 mL), and deionized water (15.58 mL) weredissolved in a separate 125 mL Erlenmeyer flask. HEMA-functionalizedbeads from Example 3 (1.0 gram) were added to the aqueous mixture andallowed to soak for 10 minutes. The beads were filtered to remove excessliquid, and then transferred into the organic toluene/heptane mixture.The suspended beads were stirred and purged with nitrogen for 30 minutesbefore adding tetramethylethylenediamine (TMEDA, 0.1 mL). The mixturewas stirred for 1 hour and then the beads were filtered. The filteredbeads were washed with acetone (3×50 mL), DI water (3×50 mL), 0.1 Nhydrochloric acid (2×50 mL), DI water (2×50 mL), acetone (3×50 ml), andthen dimethylsulfoxide (DMSO, 3×50 mL). The damp beads were placed in a50 mL polypropylene centrifuge tube and diluted up to 2× the swollenvolume with DMSO. Acetic anhydride (1.7 mL) and triethylamine (0.1 mL)were added and the mixture was mixed for 4 hours at 25° C. The beadswere filtered, washed with acetone (3×50 mL), and dried overnight underhigh vacuum. Infrared analysis indicated successful cyclization to theazlactone by the presence of an absorption band at ca. 1820 cm⁻¹.

Example 31

A Protein A coupling solution was prepared by combining 1.87 mL ofBuffer “A” (0.135 M MOPS, 1.018 M sodium sulfate, pH 7.55), 0.4 mLdeionized water, and 0.532 mL Protein A stock solution (50 mg/mL). Thissolution and a separate solution, 5.0 mL of Buffer “B” (0.100 M MOPS,0.4 M TRIS, 1.27 M sodium sulfate, pH 7.5), were equilibrated by meansof a water bath to 25° C. To a 15 mL polypropylene centrifuge tube wasadded 200 mg of the dry azlactone-functional beads from Example 26,followed by 2.80 mL of the Protein A coupling solution. The resultantslurry was mixed on an orbital shaker for 15 minutes. Buffer “B” wasadded and mixing was continued for an additional hour at 25° C. The beadslurry was centrifuged at 3000 rcf for 5 minutes, the supernate wasdecanted, and ethanolamine quench buffer (5 mL, 3.0 M ethanolamine, pH9.0) was added. This mixture was mixed for 1 hour at ambienttemperature, filtered, and washed with pH 7.5 phosphate buffer (5×20mL), then stored as a 20% ethanol solution (vol/vol) in water at 10° C.The static affinity binding capacity for IgG was measured as 55 mg/mL,while the dynamic binding capacity at 10% breakthrough was determined tobe 35 mg/mL.

Example 32

Toluene (50 mL), heptane (150 mL), and Triton X-100 (500 μL) was addedto a 3-necked round bottomed flask. The mixture was purged with nitrogenwhile stirring with an overhead stirrer at room temperature.

N-acryloyl-2-methylalanine, sodium salt (AMA-Na, 1.0 grams of a 40%solids by weight in water), sodium persulfate (0.12 grams), isopropanol(17.5 mL), and deionized water (16.9 mL) were dissolved in a separate125 mL Erlenmeyer flask. HEMA-functionalized beads from Example 1 (1.0gram) were added to the aqueous mixture and allowed to soak for 20minutes. The beads were filtered to remove excess liquid, and thentransferred into the organic toluene/heptane mixture. The suspendedbeads were stirred and purged with nitrogen for 60 minutes before addingtetramethylethylenediamine (TMEDA, 0.1 mL). The mixture was stirred for2 hours, the beads were filtered, washed with acetone (3×50 mL), DIwater (3×50 mL), 0.1 N hydrochloric acid (2×50 mL), DI water (2×50 mL),acetone (3×50 ml), and then dimethylsulfoxide (DMSO, 3×50 mL). The dampbeads were placed in a 50 mL polypropylene centrifuge tube and dilutedup to 2× the swollen volume with DMSO. Acetic anhydride (1.7 mL) andtriethylamine (0.1 mL) were added and the mixture was mixed for 4 hoursat 25° C. The beads were filtered, washed with acetone (3×50 mL), anddried overnight under high vacuum. Quantitative analysis of the infraredabsorption band at ca. 1820 cm⁻¹ indicated an azlactone content of 9.5%by weight.

Examples 33-34

Example 32 was repeated, utilizing 2.5 grams and 20 grams, respectively,of AMA-Na solution, with adjustment of the amount of added deionizedwater to 16 mL and 5.5 mL respectively. Infrared analysis of theresultant grafted beads indicated azlactone contents of 14.2% and 27.2%by weight, respectively.

We claim:
 1. An article comprising a modified grafted support of Formula(V)SS—(CO)—NH—C(R¹)₂—(CH₂)_(p)—(CO)-Q-Y¹-Q-(CO)—CR²U⁴—CH₂—U³  (V) whereinSS comprises a solid support in the form of a membrane, foam, film,sheet, or coating on a substrate; p is an integer equal to 0 or 1; eachR¹ is each independently selected from alkyl, heteroalkyl, aryl, oraralkyl; Y¹ is a first linking group comprising an alkylene,heteroalkylene, arylene, or combination thereof; each Q is independentlya divalent group selected from oxy, thio, or —NR³— where R³ is hydrogen,alkyl, heteroalkyl, aryl, or aralkyl; each R² is independently hydrogenor an alkyl; U³ comprises at least one divalent monomeric unit offormula CR²(Y²L-T)-CH₂—; Y² is a second linking group selected from asingle bond or a divalent group comprising an alkylene, heteroalkylene,arylene, or combination thereof; U³ includes at least one divalentmonomeric unit of formula —CR²(Y²L-T)-CH₂—; U⁴ is hydrogen or comprisesat least one divalent monomeric unit of formula —CR²(Y²L-T)-CH₂—; L isan attachment group formed by reacting group Z¹ with a modifying group Aof a modifying agent A-T; Z¹ is a functional group selected from (1) anacidic group or a salt thereof, (2) an amino group or a salt thereof,(3) a hydroxyl group, (4) an azlactone group or a precursor of theazlactone group, (5) a glycidyl group, or (6) a combination thereof; andT is a remainder of the modifying agent A-T and is equal to themodifying agent A-T minus the modifying group A.
 2. The article of claim1, wherein Z¹ is an acidic group and the modifying agent is of formula

wherein D is oxy, thio, or —NR³—; R³ is hydrogen, alkyl, heteroalkyl,aryl, or aralkyl; and each R⁴ is independently hydrogen, alkyl,heteroalkyl, aryl, or aralkyl.
 3. The article of claim 1, wherein Z¹ isa hydroxyl group and the modifying agent is of formula

wherein D is oxy, thio, or —NR³—; R³ is hydrogen, alkyl, heteroalkyl,aryl, or aralkyl; each R⁴ is independently hydrogen, alkyl, heteroalkyl,aryl, or aralkyl; and X is halo.
 4. The article of claim 1, wherein Z¹is an amino group and the modifying agent is of formula

wherein D is oxy, thio, or —NR³—; R³ is hydrogen, alkyl, heteroalkyl,aryl, or aralkyl; each R⁴ is independently hydrogen, alkyl, heteroalkyl,aryl, or aralkyl; and X is halo.
 5. The article of claim 1, wherein Z¹is an azlactone group and the modifying agent is of formula HD-T,wherein D is oxy, thio, or —NR³—.
 6. The article of claim 1, wherein Z¹is a glycidyl group and the modifying agent is of formula T-(CO)—OH orHD-T, wherein D is oxy, thio, or —NR³—.
 7. The article of claim 1,wherein the modifying agent is an affinity ligand comprising at leastone nucleophilic group.
 8. The article of claim 1, wherein the modifyingagent comprises a first group that is nucleophilic and a second groupthat is basic, acidic, or a salt thereof.
 9. The article of claim 1,wherein the modifying agent comprises a first group that is nucleophilicand a second group that is hydrophobic.
 10. The article of claim 1,wherein the modifying agent comprises a first group that is nucleophilicand a second group that is metal-chelating.
 11. The article of claim 1,wherein the modifying agent is a biomolecule.
 12. The article of claim1, wherein each Q is oxy.
 13. The article of claim 1, wherein Y¹ is analkylene.